Human mesenchymal stem cells (hMSCs) and their secreted extracellular vesicles (EVs) are promising therapeutics to treat degenerative or inflammatory diseases such as ischemic stroke and Alzheimer’s disease (AD). hMSC-EVs have the coveted ability to contain therapeutically relevant biomaterials; however, EV biogenesis is sensitive to the culture microenvironment in vitro. Recently, the demand for hMSC-EVs has increased dramatically, highlighting the need for scalable bioreactors for large-scale biomanufacturing. In this study, adipose-derived hMSCs were seeded in 2D plates, an ultralow-attachment (ULA) plates as static aggregates, a novel vertical wheel bioreactor (VWBR) as aggregates, and a spinner flask bioreactor (SFB). EV secretion was quantified and compared using ExtraPEG-based ultracentrifugation and nanoparticle tracking analysis. Compared to the 2D group, significantly higher total EV production and cell productivity in the bioreactors were observed, as well as the upregulation of EV biogenesis genes. Furthermore, there was increased EV production in the VWBR compared to the SFB and the static ULA control. Functional assessments demonstrated that EVs, when delivered via culture medium or hydrogel-based systems, significantly attenuated oxidative stress elevation, suppressed proinflammatory cytokine secretion (e.g., TNF-α) and gene expression, and inhibited nuclear factor kappa-light-chain-enhancer of activated B-cell (NF-κB) activation and neurodegenerative markers across in vitro assays. These findings suggest EV-mediated mitigation of oxidative and inflammatory pathways, potentially through modulation of the NF-κB signaling cascade. This study shows the influence of bioreactor types and their microenvironments on EV secretion in hMSCs and their applications in hMSC-EV production and bioengineering.

Human induced pluripotent stem cells (hiPSCs) can differentiate into various types of central nervous system organoids which are valuable for applications in tissue engineering and injury repair. The secreted extracellular vesicles (EVs) of organoids, in particular the small‐sized EV subset referred as exosomes (30–200 nm), have emerged as novel therapeutics in regenerative medicine. This study investigated the encapsulation and controlled release of human spinal cord organoid (hSCO)‐derived EVs in viscoelastic hyaluronic acid (HA) hydrogels and assessed their impact on organoid patterning. A series of pH‐responsive hydrogels were fabricated, leading to sustained EV release regulated by viscoelastic properties. The pH of these hydrogels decreased from 9 to 7 during incubation, which altered hydrogel viscoelasticity, thereby modulating EV release kinetics. In addition, EV‐loaded hydrogels regulated key hSCO patterning markers such as DBX1 and ISL1 . Furthermore, these EVs in hydrogels can cross a modeled blood–spinal cord barrier and provide cross‐barrier capability for delivery. Taken together, the organoid‐secreted EVs in viscoelastic HA hydrogels can be released at a controlled rate and have potential to regulate spinal cord organoid patterning. This study advances our knowledge of regulating intercellular communication and developing EV‐based therapies for treating neurological disorders such as spinal cord injury.